【作者】 傅思武；

【导师】 周殿元；

【作者基本信息】 第一军医大学 ， 内科学， 2004， 博士

【摘要】 艰难梭菌(C.difficile)是人与动物伪膜性肠炎(PMC)和抗生素相关性腹泻的主要病原菌，该菌可产生至少2种毒素，A毒素为肠毒素，可导致肠道组织损伤、使家兔产生血性积液并具有纤维细胞毒性，B毒素对许多组织细胞都具有很强的毒性。目前，对艰难梭菌在腹泻和结肠炎中的致病作用已有许多研究成果，而且也建立了一系列相应的治疗方法，然而，由于艰难梭菌感染的治疗较为棘手，每年仍然有数百万病人被感染，从而对艰难梭菌感染的诊断和治疗不断提出新的问题和挑战。因此，本课题在艰难梭菌A毒素纯化、单克隆抗体制备、ELISA诊断试剂的研制以及微生态制剂对艰难梭菌感染的防治方面进行了研究和探索。 首先，我们建立了一种改进的纯化艰难梭菌A毒素的方法。艰难梭菌VPl10463菌株经透析培养，50％饱和硫酸铵盐析，酸沉淀，再经DEAE-Toyopearl 650M离子交换层析。经生物学和免疫学特性分析，精制的艰难梭菌A毒素蛋白含量为0.881mg／ml，Native-PAGE及免疫双扩散均为单一带，分子量为550kD，小鼠毒力为1×10~6 MLD／ml，Vero细胞毒性为10~7CU／ml，凝血活性为1：512，家兔肠袢试验为重量长度(g／cm)比为2.46。该纯化方法操作简便、实用、适合大规模制备A毒素。 其次，成功制备了抗艰难梭菌A毒素的单克隆抗体。用纯化的艰难梭菌A毒素免疫BALB／C小鼠，将免疫小鼠的脾细胞与骨髓瘤细胞Sp2／0融合，采用间接ELISA和有限稀释法筛选和克隆化杂交瘤细胞，得到6株(2H7，3E9，485，5C10，6G8和8Al)能分泌针对艰难梭菌A毒素抗体的杂交瘤细胞株。特性分析表明，5C10株细胞分泌的mAb为IgG2a，4B5和8Al株细胞分泌的mAb为IgGl，其他3株细胞mAb(2H7、3E9和6G8)均分泌IgM。腹水mAb的效价均在1：10~4以上。中和试验表明所有的mAb均无中和活性。其中mAbZH7、6G8、5C10、4B5和8AI具有共同的表位，而mAb 3E9识别的位点与其它5株不同。InAb8AI和4B5的相对亲和力>105，其它4株mAb的相对亲和力>l护。在非变性条件下，PAGE后Westem blot的结果显示，6株mAb均可与Mr为550切的A毒素产生反应;而在变性条件下，还原与非还原sDs-P AGE后westem blot均显示，6株mAb均可与Mr为50kD一240kD的A毒素产生反应。 第三，利用该单克隆抗体，我们建立了一种具有良好特异性和灵敏度的检测艰难梭菌A毒素的夹心ELISA试剂盒。该试剂盒采用纯化的抗艰难梭菌A毒素的兔单特异血清作为捕捉抗体包板，10%BsA一PBsT封闭，加入检测样品，洗涤后加入辣根过氧化物酶标记的单克隆抗体(H即一4B5 mAb)作为检测抗体，用底物TMB显色，最后用2 mol几HZSO4终止反应，测定A450。经过对2株艰难梭菌产毒株、2株艰难梭菌非产毒株、26株大肠杆菌、4株痢疾杆菌、l株双歧杆菌、5株霍乱弧菌、2株伤寒沙门菌、7株肉毒梭菌、1株索氏梭菌、1株酪酸梭菌的培养滤液和58份粪便样品进行检测，除2株艰难梭菌产毒株的培养滤液和4份粪便样品为阳性外，其余49份培养滤液及54份粪便样品均为阴性，最低可检出艰难梭菌A毒素1呵ml。初步表明这种艰难梭菌A毒素的单克隆抗体酶标诊断试剂盒具有较高的特异度和灵敏度。可望适合临床应用。 医院内感染已经成为控制艰难梭菌感染的一个难题。根据近年来对艰难梭菌医院内感染的调查，这种感染的发生率逐年增加，说明现有的控制艰难梭菌感染的方法效果欠佳，有必要探索更好的预防和治疗方法。 最后，本课题对艰难梭菌感染后小鼠的肠粘膜损伤状况及非产毒株和酪酸梭菌对艰难梭菌感染的防治效果进行了观察。先用艰难梭菌产毒株人工感染BALB/C小鼠，感染前后分别用艰难梭菌非产毒株及酪酸梭菌进行预防与治疗，并检测盲肠内容物细胞毒性和对肠粘膜病理观察。结果证实:艰难梭菌非产毒株及酪酸梭菌不能预防BALB/C小鼠艰难梭菌的感染，但在艰难梭菌感染后使用则能明显降低艰难梭菌的产毒力和盲肠粘膜的病理损伤。说明艰难梭菌非产毒株及酪酸梭菌对小鼠艰难梭菌感染有明显的治疗作用，而且这种作用与艰难梭菌产毒降低有关。更多还原

【Abstract】 Clostridium difficile is one major causative agent of pseudomembranous colitis (PMC) and many cases of antibiotic-associated diarrhea in humans and animals. It has been reported that this organism produces at least two toxins, designated A (enterotoxin) and B (cytotoxin). Toxin A, a tissue-damaging enterotoxin, causes hemorrhagic fluid accumulation in rabbit ileal loops and is cytotoxic for cultured fibroblasts. Toxin B is an extremely potent cytotoxin for many cultured cells. Although considerable advances have been made in our understanding of the pathophysiology of diarrhea and colitis caused by C. difficile infection, and a wide variety of treatments are now available, this tenacious organism continues to infect millions of patients each year and still poses a diagnostic and therapeutic challenge.This paper presents narrower yet significant aspects of an established method for purification of C. difficile toxin, preparation of mAb of C. difficile toxin A, development of an ELISA kit for Clostridium difficile toxin A. We also studied on the preventive and curative effects of probiotics against intestinal infection of mice caused by toxigenic C. difficile.In this report, we first present a modified method for purification of toxin A. Toxingenic C. difficile VPI10463 filtrate was cultured anaerobically by the dialysis bag methods. The toxin A was then purified by precipitation with 50% (NH4)2SO4 and acid precipitation at pH5.5, followed by ion-exchange chromatography on DEAE-Toyopearl 650M. The purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis4(native-PAGE) and Ouchterlony double immunodiffusion, the molecular weight of toxin A was estimated to be 550 kD. The highly purified toxin A was characterized by its biological and immunological properties: protein concentration of 0.881mg/ml, the minimum lethal dose was IxlO6 MLD/ml(i.p.mice), the cytotoxic titer was 107CU/mg, the haemagglutination activity was at a concentration of 1:512, and the ratio of fluid weight (g) accumulated to the length (cm) of the rabbit loop was 2.46. This modified method for purification of toxin A of C. difficile was simple and convenient and offer a successful approach to produceing large volumes of toxin A from C. difficile.Monoclonal antibodies against C. difficile toxin A were then prepared. BALB/c mice were immunized with the purified C. difficile toxin A and the splenocytes from immunized mice were fused with myeloma cells Sp2/0. The hybridoma cells were screened with indirect ELISA and limiting dilution method. Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting monoclonal antibodies against C. difficile toxin A were obtained. The Ig subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8AI were IgGl, and mAb 5C10 was IgG2a. All 6 mAbs had no neutralization activity. Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9. Relative affinities of mAbs 8A1 and 4B5 were all above 105, and those of other 4 mAbs were 104. Western blot analysis after no-denatured PAGE showed that all 6 mAbs reacted to C. difficile toxin A with Mr being 550 kD, and under the of denatured condition SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C. difficile toxin A with Mr being 50 kD~240 kD.Thirdly, using the monoclonal antibodies to C. difficile toxin A, an ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. An indirect sandwich ELISA was described. After the polystyrene microtitre plates with 96 flat-bottomed well were coated with the purified rabbit mono-specific antiserum as capturing antibody, the wells were blocked with 10% BSA in PBS-T. C.?5 ?difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-Horseradish peroxidase conjugate(HRP-4B5 -mAb) was added as detecting antibody. Tetramethylbenzidines substrate was then added to each well, and the reactions were stopped by the addition of 2 mol/L sulfuric a更多还原